CN109824561B - Extraction and detection method of L-cysteine in rice seedling tissue - Google Patents
Extraction and detection method of L-cysteine in rice seedling tissue Download PDFInfo
- Publication number
- CN109824561B CN109824561B CN201910090501.6A CN201910090501A CN109824561B CN 109824561 B CN109824561 B CN 109824561B CN 201910090501 A CN201910090501 A CN 201910090501A CN 109824561 B CN109824561 B CN 109824561B
- Authority
- CN
- China
- Prior art keywords
- cysteine
- grinding
- extracting
- rice seedling
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for extracting and detecting L-cysteine in rice seedling tissues, which comprises the following steps: s1, sampling: taking rice seedling leaves or root system tissues, adding liquid nitrogen, and grinding into powder; s2, extraction: weighing 0.1-0.2 g of the powder prepared in the step S1, adding 1.0-5.0 ml of glacial acetic acid solution, and grinding for 5-10 min; sequentially adding 0.5-2.0 ml of trichloroacetic acid solution and 0.5-2.0 ml of ethanol solution, and soaking for 1-5 min to obtain a grinding fluid mixture; s3, collecting: and (4) centrifuging the grinding liquid mixture obtained in the step (S2) to obtain a supernatant, namely obtaining an extracting solution containing the L-cysteine. Adding hexadecyl trimethyl ammonium bromide and 1-chloro-2, 4-dinitrobenzene into the extracting solution, reacting at a constant temperature of 90-110 ℃, cooling after the reaction is finished, and measuring the content by a colorimetric analysis method or an absorbance photometry.
Description
Technical Field
The invention relates to the technical field of plant extraction, in particular to an extraction method and a detection method of L-cysteine in rice seedling tissues.
Background
L-cysteine is a common amino acid widely existing in animals and plants, and is widely present in a plurality of glutathione and proteins. The L-cysteine has the function of relieving and repairing the damage of radiation to human bodies, is an important nutrient component for maintaining the normal functions of livers, and has good detoxification function on toxic substances such as formaldehyde, acetaldehyde, lead, cadmium and the like. Therefore, in the daily diet, ensuring that a certain amount of L-cysteine is taken every day is of great significance to the health of the human body. The rice is staple food for 60% of people in China, and if the content of L-cysteine in the rice can be increased, the rice has important significance for improving the health of the people in China, especially for the rice in a seedling stage, if the content of L-cysteine is found to be low in time, a corresponding fertilizer can be applied as early as possible, and the content of L-cysteine in grains can be increased.
To improve the L-cysteine content in rice seedlings, L-cysteine must first be extracted from rice seedling tissues. In the prior art, the extraction and determination of L-cysteine mainly focus on the field of animal tissue research, and the extraction and determination of L-cysteine in plant tissues are relatively less. Since animal tissues and plant tissues are greatly different in various aspects such as cell structures, morphologies and component differences, the method for extracting and measuring animal tissues cannot be directly transferred to plant tissues. In addition, each plant is different from other plants, and the extraction scheme of L-cysteine in rice seedlings must be specifically found out.
After extraction, the content of L-cysteine needs to be detected, and because L-cysteine can exist stably only in a slightly acidic solution and has unstable characteristics in a neutral or slightly alkaline solution, oxidation-reduction reaction is easy to occur and interconversion with cystine is generated, the pH value of the reaction environment needs to be controlled well in the extraction and detection processes. At present, in the fields of biology and medicine, most of L-cysteine is derived to be processed by an amino acid analyzer or a high performance liquid chromatograph, but the sample pretreatment in the detection process is complex, and the detection instrument and equipment are expensive, so that the L-cysteine is difficult to be used for field detection and large sample amount detection in the rice research process.
The colorimetric detection method is a relatively common rapid detection means, L-cysteine is one of a few sulfur-containing alpha-amino acids, and the color reaction is relatively interfered to a greater extent because the L-cysteine is dark red when carrying-SH and is exposed to sodium nitroferricyanide, but the solution of the sodium nitroferricyanide is dark red.
Based on the method, the method for extracting and detecting the L-cysteine in the rice seedling tissue has important significance.
Disclosure of Invention
The first technical problem to be solved by the invention is: provides a method for extracting L-cysteine from rice seedling tissues with good extraction effect.
The second technical problem to be solved by the present invention is: provides a method for detecting L-cysteine in rice seedling tissues with obvious color change and high detection efficiency.
In order to solve the first technical problem, the invention adopts the technical scheme that: a method for extracting L-cysteine from rice seedling tissues comprises the following steps:
s1, sampling: taking rice seedling leaves or root system tissues, adding liquid nitrogen, and grinding into powder;
s2, extraction: weighing 0.1-0.2 g of the powder prepared in the step S1, adding 1.0-5.0 ml of glacial acetic acid solution, and grinding for 5-10 min; sequentially adding 0.5-2.0 ml of trichloroacetic acid solution and 0.5-2.0 ml of ethanol solution, and soaking for 1-5 min to obtain a grinding fluid mixture;
s3, collecting: and (4) centrifuging the grinding liquid mixture obtained in the step (S2) to obtain a supernatant, namely obtaining an extracting solution containing the L-cysteine.
Further, in the step S2, the volume percentage of acetic acid in the glacial acetic acid solution is 1-2%; the volume percentage of trichloroacetic acid in the trichloroacetic acid solution is 1-2%; the volume percentage of ethanol in the ethanol solution is 70-95%.
Further, in the step S2, the volume ratio of the trichloroacetic acid solution to the ethanol solution is 1 (1-2).
Furthermore, the extraction method also comprises the operation of impurity removal before sampling, namely, after cleaning the rice seedlings, absorbing the residual water on the rice seedlings by using absorbent paper.
In order to solve the second technical problem, the invention adopts the technical scheme that: a method for detecting L-cysteine in rice seedling tissues comprises the following steps:
adding hexadecyl trimethyl ammonium bromide and 1-chloro-2, 4-dinitrobenzene into the extracting solution obtained by the extracting method, reacting at a constant temperature of 90-110 ℃, reducing the temperature of the solution to 50-60 ℃ after the reaction is finished, and measuring the content of L-cysteine in the extracting solution by a colorimetric analysis method or an absorption spectrophotometry method.
Further, the extract was passed through a 0.22 μm aqueous phase filter before colorimetric detection.
Further, the cetyltrimethylammonium bromide is added in the form of an aqueous cetyltrimethylammonium bromide solution.
Further, the 1-chloro-2, 4-dinitrobenzene is added in the form of an ethanol solution of 1-chloro-2, 4-dinitrobenzene.
Further, the isothermal reaction is completed in a boiling water bath environment.
The invention has the beneficial effects that: the scheme of the invention firstly provides the extraction and determination of L-cysteine from rice, and the detection scheme of the invention is combined with detection, so that the high-efficiency and rapid detection can be realized, the total time from extraction to detection is not more than 30 min. Compared with the existing literature data, the amino acid analyzer analysis method and the derivative-high performance liquid chromatography analysis method have the advantages that the time consumption is obviously shortened, complex equipment is not needed, the whole extraction and detection process can be operated on the field of rice test fields, and in addition, reagents in the whole process can be prepared into a kit for industrial batch production.
Drawings
FIG. 1 is a graph showing the relationship between concentration and absorbance measured by UV spectrophotometry in example 1 of the present invention;
FIG. 2 is a liquid chromatogram measured in comparative example 1 of the present invention.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The embodiment 1 of the invention is as follows: a method for extracting L-cysteine from rice seedling tissues comprises the following steps:
(1) taking Hunan early indica No. 45 (purchased from agricultural academy of sciences of Hunan province) rice seedlings cultivated by a sand culture method for 15 days, cleaning sand grains remained on root systems of the rice seedlings by using clear water, then sucking excessive water on the root systems, and slightly pressing the rice seedlings without breaking the roots and leaves when absorbing water.
(2) And (3) selecting and weighing the rice leaf or root system tissue obtained in the step (1), and quickly grinding the rice leaf or root system tissue into uniform powder by using liquid nitrogen in a mortar.
(3) Three groups of 0.13g, 0.11g and 0.13g rice seedling leaf tissue powders were weighed out, respectively.
(4) Adding 1% glacial acetic acid (to destroy fiber structure in tissue) 4.0mL, grinding for 5min, sequentially adding trichloroacetic acid 1.0mL and ethanol 95% (to remove macromolecular protein and precipitate nucleic acid) 1.0mL, soaking for 2min to obtain grinding mixture.
(5) And (4) transferring the grinding fluid mixture obtained in the step (4) into a plastic centrifuge tube, centrifuging for 10 minutes at 10000rmp at 4 ℃, and taking supernatant.
(6) And (4) filtering the supernatant obtained in the step (5) by a 0.22-micron water-phase filter membrane, and placing the filtrate in a refrigerator at 4 ℃ for analysis.
(7) Accurately adding 10 mu L of 0.10mol/L hexadecyl trimethyl ammonium bromide aqueous solution and 1 mu L of 0.07mol/L ethanol solution of 1-chloro-2, 4-dinitrobenzene into the filtrate, and quickly placing the mixture into a water bath at 100 ℃ for 2min (or prolonging the heating time to 5-8 min).
(8) After the boiling water bath is finished, the temperature is reduced to between 50 and 60 ℃. The absorbance of the sample was recorded using an ultraviolet and visible spectrophotometer with the wavelength set at 355 nm. A blank reference solution was selected at zero concentration, i.e.without L-cysteine (otherwise identical to the treatment of the extract).
(9) In the UV and visible spectrophotometer analysis, if the absorbance value of the sample exceeds 0.8, the sample needs to be diluted. The test predicts that the absorbance value reaches more than 2.5, the samples are diluted by 10 times before measurement, and the measurement results are shown in the following table 1:
TABLE 1 measurement of experimental data by UV spectrophotometry
| Sample numbering | Leaf weight (g) | Absorbance of the solution | Concentration (mmol/L) |
| 1 | 0.13 | 0.254 | 0.129 |
| 2 | 0.11 | 0.240 | 0.122 |
| 3 | 0.13 | 0.242 | 0.123 |
By measuring the L-cysteine in the leaf part of the rice, the RSD of the parallel sample is 0.3 percent, and the concentration is 0.122-0.129, so that the method is stable and reliable.
(10) Ultraviolet detection linear range and detection limit determination: preparing a series of solutions with L-cysteine concentrations of 50 mu mol/L, 40 mu mol/L, 30 mu mol/L, 20 mu mol/L, 10 mu mol/L and 0 mu mol/L, repeating the operations in the steps (7) to (8), measuring the absorbance values, finding that the linear relation is good in the range of 0-40 mu mol/L, drawing the absorbance and the corresponding concentration points, and then performing linear fitting, wherein the fitting linear equation is y-0.02 x-0.0866, as shown in FIG. 1, and the specific fitting information is shown in the following table 2:
TABLE 2 Linear fitting parameters
As can be seen from the above table, the linear relationship is good in the range of 1 to 40. mu. mol/L, and the lowest detection limit is 0.5. mu. mol/L.
Comparative example 1
L-cysteine can also be determined by high performance liquid chromatography, and because cysteine has larger polarity, the L-cysteine is generally subjected to derivatization by a derivatization agent and then is subjected to sample analysis.
The procedure for obtaining the supernatant by grinding rice was the same as in the procedures (1) to (5) of example 1.
The liquid chromatography conditions used in this example are as follows:
the agent 1260 was fitted with a C18 column (250mm x 4.6mm i.d., 5 μm) with a fluorescence detector (excitation wavelength 470nm, emission wavelength 540 nm).
Gradient elution procedure:
a: methanol, B: acetonitrile, C: pure water, D: 0.01mol/L phosphate buffer solution.
1，0-5min，6.5％A，6.5％B，C87％，D1％；
2，5-15min，25％A，25％B，C49％，D1％；
3，15-20min，35％A，35％B，C29％，D1％；
The chromatogram after the sample injection analysis of L-cysteine with the same concentration is shown in FIG. 2, and it can be seen from FIG. 2 that the high performance liquid chromatography is adopted, although L-cysteine can be detected, the operation is complicated and the time consumption is long.
Three samples were also measured and the results are shown in table 3 below:
TABLE 3 measurement results of chromatography
| Sample numbering | Leaf weight (g) | Concentration (mmol/L) |
| 1 | 0.14 | 0.135 |
| 2 | 0.13 | 0.120 |
| 3 | 0.13 | 0.128 |
From the above results, it can be seen that the results obtained using the protocol of the present invention are substantially identical to those obtained using chromatography.
Comparative example 2
The only difference from example one is that sulfuric acid is added before grinding in step (4).
The step (4) is specifically as follows: adding 1% sulfuric acid 4.0mL, grinding for 5min, sequentially adding trichloroacetic acid 1.0mL and 95% ethanol 1.0mL, and soaking for 2min to obtain grinding liquid mixture.
As a result: the color development time is as long as 1 hour, and the light absorption value is unstable.
Comparative example 3
The only difference from example one is that formic acid was added before grinding in step (4).
The step (4) is specifically as follows: (4) adding 1% sulfuric acid 4.0mL, grinding for 5min, sequentially adding trichloroacetic acid 1.0mL and 95% ethanol 1.0mL, and soaking for 2min to obtain grinding liquid mixture.
As a result: no color development and no detection.
Comparative example 4
The only difference from example one is that in step (4), ethanol is added first and then trichloroacetic acid is added.
The step (4) is specifically as follows: (4) adding 1.0mL of 95% ethanol and 4.0mL of 1% glacial acetic acid, grinding for 5min, sequentially adding 1.0mL of 1% trichloroacetic acid, and soaking the solution for 2min to obtain a grinding fluid mixture.
As a result: no color development and no detection.
The pretreatment treatment process of the plant tissue sample is simple and convenient to operate, the color development time in the analysis process is short, the effect is stable and reliable, the dosage of the color developing agent is small, and the rapid detection of the L-cysteine in the plant tissue is realized.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (4)
1. A method for extracting L-cysteine from rice seedling tissues is characterized by comprising the following steps: the method comprises the following steps:
s1, sampling: taking rice seedling leaves or root system tissues, adding liquid nitrogen, and grinding into powder;
s2, extraction: weighing 0.1-0.2 g of the powder prepared in the step S1, adding 1.0-5.0 ml of glacial acetic acid solution, and grinding for 5-10 min; sequentially adding 0.5-2.0 ml of trichloroacetic acid solution and 0.5-2.0 ml of ethanol solution, and soaking for 1-5 min to obtain a grinding fluid mixture;
s3, collecting: and (4) centrifuging the grinding liquid mixture obtained in the step (S2) to obtain a supernatant, namely obtaining an extracting solution containing the L-cysteine.
2. The method for extracting L-cysteine from a rice seedling tissue according to claim 1, wherein: in the step S2, the volume percentage of acetic acid in the glacial acetic acid solution is 1-2%; the volume percentage of trichloroacetic acid in the trichloroacetic acid solution is 1-2%; the volume percentage of ethanol in the ethanol solution is 70-95%.
3. The method for extracting L-cysteine from a rice seedling tissue according to claim 1, wherein: in the step S2, the volume ratio of the trichloroacetic acid solution to the ethanol solution is 1 (1-2).
4. The method for extracting L-cysteine from a rice seedling tissue according to claim 1, wherein: the extraction method also comprises the operation of impurity removal before sampling, namely, after cleaning the rice seedlings, absorbing the residual water on the rice seedlings by using absorbent paper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910090501.6A CN109824561B (en) | 2019-01-30 | 2019-01-30 | Extraction and detection method of L-cysteine in rice seedling tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910090501.6A CN109824561B (en) | 2019-01-30 | 2019-01-30 | Extraction and detection method of L-cysteine in rice seedling tissue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109824561A CN109824561A (en) | 2019-05-31 |
| CN109824561B true CN109824561B (en) | 2020-09-22 |
Family
ID=66862953
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910090501.6A Active CN109824561B (en) | 2019-01-30 | 2019-01-30 | Extraction and detection method of L-cysteine in rice seedling tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109824561B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1486978A (en) * | 2003-08-08 | 2004-04-07 | 华侨大学 | L-cystine extracting process |
| CN103969253A (en) * | 2013-07-23 | 2014-08-06 | 中国科学院烟台海岸带研究所 | Method for simply rapidly detecting L-cysteine |
| CN106565565A (en) * | 2016-10-19 | 2017-04-19 | 武汉远大弘元股份有限公司 | Preparation method of carbocisteine |
| CN109283146A (en) * | 2018-09-19 | 2019-01-29 | 天津工业大学 | Sensor and detection method for L-cysteine detection |
-
2019
- 2019-01-30 CN CN201910090501.6A patent/CN109824561B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1486978A (en) * | 2003-08-08 | 2004-04-07 | 华侨大学 | L-cystine extracting process |
| CN103969253A (en) * | 2013-07-23 | 2014-08-06 | 中国科学院烟台海岸带研究所 | Method for simply rapidly detecting L-cysteine |
| CN106565565A (en) * | 2016-10-19 | 2017-04-19 | 武汉远大弘元股份有限公司 | Preparation method of carbocisteine |
| CN109283146A (en) * | 2018-09-19 | 2019-01-29 | 天津工业大学 | Sensor and detection method for L-cysteine detection |
Non-Patent Citations (2)
| Title |
|---|
| L-半胱氨酸的生产方法及应用进展;贾存江等;《齐鲁药事》;20071224;第26卷(第9期);P553-555 * |
| 硫对水稻幼苗乙烯产生的影响;张英聚等;《植物学报》;19891231;第31卷(第10期);P775-779 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109824561A (en) | 2019-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103091422B (en) | Method for detecting residual quantity of sodium nitrophenol in aquatic product | |
| CN106610410B (en) | Detection method of biogenic amine in fish and products thereof | |
| CN105052576B (en) | A kind of determination method of protected crop drought stress grade | |
| CN104231038A (en) | Extraction and purification method of decolorized cardamine hupingshanesis selenium-containing proteins | |
| CN103335990A (en) | Method for measuring methyl mercury and ethyl mercury in animal flesh | |
| Briggs et al. | Some properties of phytochrome isolated from dark-grown oat seedlings (Avena sativa L.) | |
| Dresler et al. | Capillary zone electrophoresis for determination of reduced and oxidised ascorbate and glutathione in roots and leaf segments of Zea mays plants exposed to Cd and Cu | |
| CN101696964A (en) | Solid phase extraction and HPLC-fluorescence detection method for fluoroquinolones antibiotics | |
| Rienth et al. | Validation and application of an improved method for the rapid determination of proline in grape berries | |
| CN111812229A (en) | Analysis method for measuring 2-methylbenzothiazole in soil/sediment through gas chromatography-mass spectrometry | |
| CN102081078A (en) | Method for measuring residual quantities of four fluoroquinolone medicaments in animal food | |
| CN109824561B (en) | Extraction and detection method of L-cysteine in rice seedling tissue | |
| CN113702524A (en) | Separation and analysis method for 8 arsenic species with different forms in plant body, water body and sediment body | |
| CN104122241A (en) | Method for rapid determination of selenium content in tetrastigmatis hemsleyani | |
| Sharma et al. | Fluoride mediated biochemical responses and removal potential in hydroponically grown duckweed (Spirodela polyrhiza L. scheldien) | |
| Drochioiu et al. | The breakdown of plant biostructure followed by amino acids determination | |
| CN104483275A (en) | Detection method of biothiol | |
| CN108562471B (en) | Method for rapidly detecting activity of plant root system | |
| CN112666302B (en) | Method for identifying active flavone component group in barley seedling and rapidly detecting active flavone component group | |
| Bright et al. | Amino acids in lacustrine ostracodes, part III: Effects of pH and taxonomy on racemization and leaching | |
| CN112305112B (en) | Method for identifying mint-fed grass carp and common-fed grass carp | |
| CN112129745B (en) | Method for measuring lead content in industrial hemp processing extract by microwave digestion-graphite furnace atomic absorption | |
| CN108918638A (en) | A kind of test method that chronic administration height nitrogenous fertilizer influences corn kernel albumin | |
| EP3279666A1 (en) | Means and methods for determination of isotope labeled amino acids | |
| CN103063758A (en) | Method for detecting Xanthophyll cycle components of alfalfa |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |